U.S. patent number 5,943,129 [Application Number 08/906,665] was granted by the patent office on 1999-08-24 for fluorescence imaging system.
This patent grant is currently assigned to Cambridge Research & Instrumentation Inc.. Invention is credited to Clifford C. Hoyt, Peter J. Miller.
United States Patent |
5,943,129 |
Hoyt , et al. |
August 24, 1999 |
Fluorescence imaging system
Abstract
A fluorescence imaging system for epi-illumination wherein the
usual dichroic beamsplitter is replaced by a polarizing
beamsplitter (PBS). The sample is excited with light that is
linearly polarized to a significant degree, and fluorescent
emission light is collected in the orthogonal linear polarization
state. Excitation light that is scattered or reflected by the
sample is rejected by the PBS, while the desired emission light is
captured for imaging by a detector. By eliminating the usual
dichroic beamsplitter member, the imaging system removes the
barriers to multi-spectral imaging that such members conventionally
impose. A wavelength-selective birefringent network may also be
interposed between the beamsplitter and the sample for converting
the polarization of either the excitation or the emission light to
the orthogonal state without defeating this desirable rejection
property, thus permitting measurement of the sample emissions in
either or both linear polarization states for assessing
fluorescence polarization.
Inventors: |
Hoyt; Clifford C. (Needham,
MA), Miller; Peter J. (Somerville, MA) |
Assignee: |
Cambridge Research &
Instrumentation Inc. (Cambridge, MA)
|
Family
ID: |
25422783 |
Appl.
No.: |
08/906,665 |
Filed: |
August 7, 1997 |
Current U.S.
Class: |
356/318;
250/458.1; 356/417 |
Current CPC
Class: |
G01N
21/6445 (20130101); G01N 21/6456 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G01N 021/64 () |
Field of
Search: |
;356/317,318,417
;250/458.1,459.1,461.1,461.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
"Synthesis of Optical Birefrigerent Networks", Progress in Optics
IX (North-Holland Amerstand) 1971, pp. 123-177 by E.O. Amman. .
"Flat Passband Birefrigerent Wavelength Domain Multiplexer",
Electronics Letters 23(3), 106-7 (1987) by W.J. Carlsen and C.F.
Buhrer. .
"Optical Network Synthesis Using Birefrigerent Crystals. I.
Synthesis of Lossless Networks of Equal-Length Crystals", J. Opt.
Soc. Am. 54, 1267 (1264) by S.E. Harris, E.O. Amman and C.
Chang..
|
Primary Examiner: Evans; F. L.
Attorney, Agent or Firm: Cohen, Potani, Lieberman &
Pavane
Claims
We claim:
1. A fluorescence imaging system for imaging radiation emitted by a
sample to be imaged by the system through fluorescing of the sample
in response to illumination of the sample by illuminating
radiation, said system comprising:
an illumination source operable for emitting a beam of optical
illuminating radiation along a first optical path;
a detector for receiving and imaging radiation emitted from a
sample along a second optical path in response to illumination of
the sample by the illuminating radiation;
a polarizing beamsplitter for (1) receiving illuminating radiation
from the illumination source along the first optical path and
selectively redirecting only the illuminating radiation of a first
polarization state to the second optical path for illuminating the
sample with the redirected illumination radiation of the first
polarization state so as to effect fluorescing of the sample and
emission of radiation from the sample, and for (2) receiving
emission radiation from the sample along the second optical path
and selectively transmitting through the beamsplitter along the
second optical path for receipt by said detector only the emission
radiation from the sample having substantially a second
polarization state orthogonal to the first polarization state;
and,
at least one additional polarizer disposed in at least one of the
first and second optical paths, which preferentially reduces the
proportion of excitation light incident upon the detector.
2. The system of claim 1, wherein said polarizing beamsplitter
comprises a prism-type polarizing beamsplitter cube.
3. The system of claim 1, wherein said polarizing beamsplitter
comprises a plate-type polarizing beamsplitter.
4. The system of claim 1, wherein said polarizing beamsplitter
element comprises a reflective polarizing film.
5. The system of claim 1, wherein the said least one additional
polarizer is located in the first optical path between the
illumination source and the polarizing beamsplitter for selectively
polarizing the illumination radiation for receipt by the
beamsplitter in the first polarization state.
6. The system of claim 1, wherein the said least one additional
polarizer is located in the second optical path between the
detector and the polarizing beamsplitter for further polarizing the
emission radiation transmitted by the beamsplitter.
7. The system of claim 1, further comprising an optical filter in
the first optical path between the illumination source and the
polarizing beamsplitter for transmitting optical radiation within a
selected wavelength range.
8. The system of claim 7, wherein said optical filter comprises a
tunable filter.
9. The system of claim 7, further comprising a fiber-optic element
in the first optical path between the optical filter and the
polarizing beamsplitter for transmitting optical radiation within a
selected wavelength range.
10. The system of claim 1, further comprising a filter in the
second optical path between the polarizing beamsplitter and the
detector.
11. The system of claim 10, wherein said filter comprises a tunable
filter.
12. The system of claim 1, further comprising spectrometer means
for determining spectral content of the emitted radiation incident
upon said detector.
13. The system of claim 12, wherein said spectrometer means
comprises one of a diffraction grating, a Michelson interferometer,
a Sagnac interferometer, and a Fabry-Perot interferometer.
14. The system of claim 1, further comprising at least one
birefringent element disposed in the second optical path between
the polarizing beamsplitter and the sample, which transmits the
excitation beam substantially in one of its initial polarization
state and the orthogonal polarization state.
15. The system of claim 14, wherein said at least one birefringent
element comprises a liquid crystal element.
16. The system of claim 14, further comprising mechanical means for
selectively moving said at least one birefringent element into and
out of the second optical path.
17. The system of claim 14, further comprising means for
sequentially illuminating the sample location in each of two
substantially orthogonal polarization states.
18. The system of claim 14, further comprising means for
sequentially imaging emission radiation in each of two
substantially orthogonal polarization states.
19. The system of claim 14, wherein said at least one birefringent
element comprises a plurality of birefringent elements and an
optical polarization switch having a first setting at which said
plural birefringent elements transmit radiation within a
predetermined range of wavelengths without alteration of
polarization state.
20. The system of claim 19, wherein said optical polarization
switch has a second setting at which said plural birefringent
elements convert radiation within a first selected wavelength range
from an initial polarization state to a final polarization state
orthogonal to the initial polarization state and transmit light
within a second selected wavelength range without substantial
alteration of polarization state.
21. The system of claim 1, further comprising a reflective
polarizer element disposed in the first optical path between the
illumination source and the polarizing beamsplitter.
22. The system of claim 21, further comprising means for directing
radiation reflected by said reflective polarizing element back
toward the reflective polarizing element.
23. The system of claim 22, further comprising conversion means
disposed between said reflective polarizing element and the
illumination source for converting at least a portion of the
radiation reflected by said reflective polarizing element from an
initial polarization state when reflected by the reflective
polarizing element to a final polarization state orthogonal to the
initial polarization state.
24. The system of claim 1, further comprising a twisted nematic
liquid crystal cell disposed in the second optical path between the
polarizing beamsplitter and the sample.
25. The system of claim 4, wherein the film comprises DBEF.
26. The system of claim 8, wherein the tunable filter comprises at
least one of an acousto-optical tunable filter, a liquid crystal
tunable filter, a surface plasmon filter, and a mechanical filter
wheel.
27. The system of claim 11, wherein the tunable filter comprises at
least one of an acousto-optical tunable filter, a liquid crystal
tunable filter, a surface plasmon filter, and a mechanical filter
wheel.
28. The system of claim 1 wherein the at least one additional
polarizer is a linear polarizer.
Description
BACKGROUND OF THE INVENTION
The present invention is directed to a fluorescent imaging
system.
There are currently numerous methods for fluorescent imaging, all
of which have as their objective the illumination of a sample with
excitation light of one wavelength while imaging a resulting
fluorescent emission at a second, longer wavelength. Because the
fluorescent efficiency of many samples is low, i.e. typically 1
photon of fluorescent emission or less per 100 photons of
excitation, the optical imaging system must efficiently collect the
weak fluorescent emission without interference from the much
stronger excitation signal. The optical system must provide an
efficient optical path for delivering emission light to the image,
but little or no such path for excitation light. Typically,
spectral filters, such as colored-glass or interference filters,
are used to provide at least some degree of the required wavelength
selectivity which is enhanced through a careful choice of the
overall optical design.
Prior art optical systems normally incorporate one optical path for
the excitation light and a second optical path for the fluorescent
emission. These optical paths necessarily overlap at the sample,
and in many systems the two paths also make common use of one or
more of the optical elements.
Two approaches are widely used to minimize the coupling of light
from the excitation to the emission optical path. The first
approach is to illuminate the sample with light having a range of
angles to which the emission optics are non-responsive. One method
for achieving this is illuminating the sample from the side while
collecting the fluorescent emission from the top. A portion of the
fluorescent emission, which is more or less isotropic in angular
distribution, is captured by the collection optics while the
angularly-restricted excitation beam leaving the sample proceeds,
uncollected, to a baffled optical trap.
Although such an arrangement can be used successfully for
single-point measurements, it is not generally suitable in imaging
applications. Imaging systems commonly use "dark-field"
illumination, in which diaphragms, or zone plates, in the
illumination and collection objectives insure that the sample is
illuminated over a first selected cone of angles while the emission
is collected over a second, but different, cone of angles. An
inherent feature of the dark-field method is that the effective
numerical aperture ("NA") of the objectives is reduced, causing a
significant and undesired reduction in optical efficiency.
Moreover, by reducing the NA the diffraction limit of the
instrument is degraded so that both image quality and resolution
also deteriorate.
The second known approach to minimizing coupling of light from the
excitation to the emission path is separating those two optical
paths through the use of a dichroic beamsplitter. Most widely
employed is the "epi-illumination" method, which utilizes a
dichroic beamsplitter that strongly reflects light at the
excitation wavelength, but transmits light at the emission
wavelength. The beamsplitter is oriented to reflect light from the
illumination optics into a common objective through which it
illuminates the sample. Fluorescent emission, collected by the same
objective, passes through the dichroic beamsplitter without
significant loss and proceeds along the remainder of the emission
optical path. Since the beamsplitter provides low transmission at
the excitation wavelength, little of the stray excitation light
that is reflected or scattered by the sample finds its way into the
emission optical path. Unlike the dark-field arrangement, there are
no limitations on the NA of the objective, and it is possible to
use objectives with a high NA to achieve high throughput and high
image resolution.
However, in the epi-illumination method the dichroic beamsplitter
inherently restricts one to a single set of excitation and emission
wavelengths since the beamsplitter affords high reflection at a
particular predetermined band of excitation wavelengths and high
transmission at another particular predetermined band of emission
wavelengths.
While it is possible to design a dichroic beamsplitter which
provides for three or even four excitation bands and a
corresponding number of emission bands, for several reasons such
beamsplitters offer only a limited increase in versatility. First,
the optical performance of a multi-band device is generally
inferior to that of a single-band device due to limitations in the
optical coating art. Second, the wavelengths of the various bands
cannot be independently specified or selected due to constraints in
the thin-film coating art. However, each particular fluorescent
species has a spectral response which dictates the use of an
optimal band for excitation and, accordingly, an optimal emission
band. In practice, the various bands reflected and passed by the
beamsplitter cannot all be chosen for maximum efficiency of
excitation and collection, with the result that for one or more
fluorescent species, the system is inefficient at the excitation
fluorescence and/or at the fluorescent emission signal
wavelengths.
A third drawback of multiband beamsplitters is that any given
wavelength must be dedicated to either excitation or to emission.
If dedicated to excitation, the dichroic beamsplitter must be
highly reflective, whereas for emissions it must be highly
transmissive. Thus, it is fundamentally impossible with a dichroic
beamsplitter to observe fluorescent emission at any wavelength
which is or may be used as an excitation band. This presents a
severe restriction in attempts to devise a system for imaging
multiple fluorescent species. It is undesirable to mechanically
exchange the beamsplitter to overcome this restriction because this
leads to vibration and image shift in the system.
In addition, the restrictions imposed by a dichroic beamsplitter
severely restrict spectroscopic imaging systems. When more than one
fluorescent species is present, the emission spectra may overlap
and the observations at a single wavelength may not uniquely
identify the emitting species. By taking a complete spectrum and
resolving it into relative contributions from the different
species, each of which has a characteristic spectral shape, the
presence and quantity of each species can be accurately determined.
However, this requires a fluorescence imaging system to obtain a
spectrum at many wavelengths. Although it is ideally desirable to
accommodate a continuous unbroken spectrum over the full range of
emission, such an objective is not achievable by the use of a
dichroic beamsplitter which merely dedicates specific fixed
wavelength bands to excitation and others to emission.
Thus, there is currently no fluorescent imaging system that
accommodates the use of high and unrestricted NA in the objectives
and which does not impose severe limits on the spectral location of
the excitation and emission bands that are employed for imaging
multiple fluorescent species, or from spectroscopic imaging of
fluorescent species.
SUMMARY OF THE INVENTION
It is accordingly an object of the present invention to provide a
fluorescent imaging system that can be used with many fluorescent
species and which permits dense and even overlapping spectral bands
to be employed for excitation and emission. The invention overcomes
the limitations imposed by multiband dichroic beamsplitters, and
eliminates the need for interchanging simple dichroic beamsplitters
when imaging multiple fluorescent species. The present invention
further provides a system with no moving parts for measuring
fluorescent polarization with the option of using several
excitation and emission bands.
The invention is more particularly directed to a fluorescent
imaging system in which the normal dichroic beamsplitter is
replaced by a polarizing beamsplitter (PBS). The fluorescent
imaging system of the invention includes an illumination source
that provides a beam of optical radiation along an optical path. A
PBS element, which selectively reflects or transmits optical
radiation differentially based on its polarization state, is
disposed in the optical path. The optical radiation is at least
predominantly in a first polarization state leaving the PBS element
and may contain a small amount of light orthogonal to the first
polarization state, and is directed from the PBS toward a location
at which the sample being observed is located. The emissions from
the sample pass through the PBS element and, by selective
reflection or refraction in the PBS element, are selected to be
substantially of the orthogonal polarization state to the first
polarization state. The system additionally includes a detector
that is responsive to, and is disposed to receive, fluorescent
emissions from the sample.
The illumination optics generate light that is linearly polarized
in a state for which the PBS is highly reflective. Fluorescent
emissions having the orthogonal polarization state, for which the
PBS is transmissive, are collected and used to form an image of the
sample fluorescence. Reflected excitation light having the same
polarization state for which the PBS is highly reflective is
rejected by the PBS and thus proceeds no further along the emission
optical path. The present invention accordingly provides a high
degree of selectivity between the excitation and emission light,
without the restrictions on NA that are imposed by dark-field
systems.
The various features of novelty which characterize the invention
are pointed out with particularity in the claims annexed to and
forming a part of the disclosure. For a better understanding of the
invention, its operating advantages, and specific objects attained
by its use, reference should be had to the drawing and descriptive
matter in which there are illustrated and described preferred
embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings, wherein like reference characters denote similar
elements throughout the several views:
FIG. 1 graphically depicts the absorption and emission spectra for
two fluorescent species;
FIG. 2 diagrammatically illustrates a prior-art epi-illuminated
system for fluorescent imaging;
FIG. 3 depicts the properties of a polarizing beamsplitter
(PBS);
FIG. 4 depicts an imaging system constructed in accordance with the
present invention;
FIG. 5 illustrates another embodiment of an imaging system in
accordance with the invention;
FIG. 6 graphically shows the properties of a birefringent network
for light of various wavelengths;
FIG. 7 shows another embodiment of an imaging system in accordance
with the present invention;
FIG. 8 illustrates still another imaging system in accordance with
the invention;
FIG. 9 shows a construction of a non-switchable birefringent
network;
FIG. 10 graphically illustrates the properties of a switchable
birefringent network for light of various wavelengths;
FIGS. 11a and 11b depict a suitable switch, in its respective "off"
and "on" states, for use in constructing a switchable birefringent
network;
FIG. 12 illustrates a construction of a switchable birefringent
network;
FIG. 13 depicts yet another embodiment of a fluorescent imaging
system of the invention incorporating a switchable birefringent
network;
FIG. 14 shows still another embodiment in accordance with the
invention in which the illumination system incorporates a
fiber-optic feed;
FIG. 15 depicts another embodiment of the present invention in
which the imaging system incorporates a tunable filter or
spectrometer in the emission path;
FIG. 16 shows an imaging system in accordance with the invention in
which the system incorporates a filter wheel or tunable filter in
the illumination path; and
FIG. 17 shows still another imaging system of the invention in
which the system incorporates one or more reflective polarizer
elements located between the polarizing beamsplitter and other
components of the system.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring initially to FIG. 1, a first sample species has an
absorption spectrum (curve 2) with maximum absorption at a
wavelength identified as a spectral feature 3 and an emission
spectrum, depicted by curve 4, when excited at the wavelength 3.
The emission spectrum 4 shows a maximum near the wavelength
identified as 5. A second species has an absorption spectrum (curve
6) with maximum absorption at a wavelength identified as spectral
feature 7. When excited at the wavelength 7, the emission spectrum
is shown by curve 8; this emission has a maximum at about a
wavelength identified as 9.
FIG. 2 depicts a prior art fluorescent imaging system. Light from
an illumination source 150 passes through associated illumination
optics 151 and then to a first spectral filter 152. Filter 152
transmits light 154 of a selected spectral range to excite
fluorescence. Excitation light 154 proceeds to a dichroic
beamsplitter 153 that reflects the light 154 toward objective 156,
at which the rays 157 are focused on a sample 160. Fluorescent
emission 158 from the sample is gathered by the objective 156 and
passes through dichroic member 153 to a second spectral filter 162
that transmits light 161 within a selected spectral range.
Image-forming optics 163 form an image 164 of the fluorescent
emission upon a detector 165.
FIG. 3 illustrates the properties of a polarizing beamsplitter
(PBS) element; the figure presents a side-view in which all rays
(except as otherwise indicated) lie in the plane of the drawing. As
shown in FIG. 3, a beam of light 30 is incident on a PBS 35. The
beam 30 consists of optical radiation having a polarization mode 31
with an E-field in the plane of the drawing, and a polarization
mode 32 having an E-field perpendicular to the plane of the
drawing. By convention, components 31 and 32 are respectively
referred to herein as the P component and the S component. At PBS
element 35, the S component 32 is substantially reflected and
becomes component 36 of beam 33 with a polarization E-field that
remains perpendicular to the plane of the drawing. A weak component
is transmitted by the PBS 35 and becomes component 37 of a beam 34.
Conversely, the P-component 31 is substantially transmitted through
PBS 35, and only a weak portion is reflected to form component 38
of beam 33 having an E-field in the plane of the drawing; the major
portion of P-component 31 is transmitted through PBS 35 and becomes
component 39 of beam 34. Although the PBS is shown as a cube with
its hypotenuse at a 45.degree. angle to the incident beam 30, the
PBS may alternatively comprise a plate-type or reflective
polarizing film-type PBS device and nevertheless operate in a
similar fashion. In addition, the PBS devices are operable at
alternative incidence angles other than 45.degree..
FIG. 4 depicts a first embodiment of a system implementing the
invention. An illumination source 11 provides optical radiation,
and may be any suitable light source including by way of
illustrative example a conventional light bulb, a laser, an arc
lamp, a krypton lamp, a xenon lamp and a pulsed flash tube. The
optical radiation from source 11 travels along an optical path in
which associated optics 12 are located. The optical radiation
passes through the optics 12 to a first spectral filter 14 that
transmits light of a selected spectral range to a polarizing
beamsplitter element 15, the functioning of which has been
described in connection with FIG. 3. The selected light is thus
reflected by PBS 15 and thereby directed to an objective 16, which
further directs and focuses the light onto a sample 20 that is
capable of responsively fluorescing and emitting light when so
illuminated. A portion of the light emitted from the sample 20 is
selectively passed by PBS 15 and is thereby delivered to imaging
optics which include a second spectral filter 22 and image forming
optics 23 that collect the fluorescent emissions from sample 20 to
form an image 24 on a detector 25.
The present invention will find application in numerous fields such
as imaging science, microscopy, optical design, birefringent
filters, and tunable filters. The inventive system is especially
suitable for use in many areas in which imaging is employed, such
as drug assays, genetic or DNA analysis and fluorescent
imaging.
FIG. 5 depicts a modified form of the inventive system that
additionally incorporates an optical filter 41 in the path of the
imaging optics to provide additional rejection of any stray or
scattered excitation light and thereby prevent such strong light
from unintentionally contributing to the image developed at
detector 25.
The wavelengths of optical radiation that are used for the
excitation and emission bands may be selected for reflection and
transmission within the system by the use, for example, of fixed
filters, a filter wheel, or tunable filters such as acousto-optic
tunable filters ("AOTF") or liquid crystal tunable filters
("LCTF"). A large number of independently chosen wavelength bands
may be employed for excitation and for responsive emission with
either type of tunable filter. Since the polarizing action of the
PBS is effective over a wide spectral range, a similarly wide
spectral range may be employed in the overall system. Moreover,
inasmuch as the excitation and emission wavelengths may be
independently selected, a system which collects emission light at a
given wavelength when imaging one fluorescent species and then,
after adjustment of the excitation and emission filters, uses this
same wavelength for sample excitation can be constructed. It is not
necessary to mechanically change the beamsplitter elements, even if
the bands used at different times for excitation overlap with the
bands used at different times for emission. For each selected
excitation wavelength, a complete emission spectrum can be obtained
over a broad range free of gaps or inaccessible spectral bands. The
present invention is therefore advantageously suitable for use with
spectral instruments to obtain spectra of fluorescent emission.
Examples of such equipment include, without limitation, grating
spectrometers, Michelson interferometers, Sagnac interferometers,
and tunable filters such as AOTFs and LCTFs.
As noted above, the PBS may be operated at incidence angles
.theta..sub.inc other than 45.degree.. If operated in this manner,
the angle through which the excitation light is reflected by the
PBS will not be 90.degree., but another angle which is given by
2.theta..sub.inc. Also, while the optical system in FIG. 4 shows
the excitation and emission beams perfectly oriented or
coincidental with the optical axis of objective 16, it is possible
to construct the system of the invention with excitation and
emission beams oriented so their rays do not coincide with the
optical axis of this objective. The off-set may be a small
difference which arises as a consequence of manufacturing
tolerances in constructing the system, or it may be an intentional
difference of several degrees up to about 5.degree.. Such a design
may be utilized, if the objective has sufficient field-of-view, to
reduce normal-incidence Fresnel reflections which would otherwise
occur from the sample such as e.g. the glass surface of a
microscope slide.
If a filter wheel is used to select the wavelength band of
excitation light, the light source and filter wheel may be located
separately from the remainder of the system and the excitation
light may be introduced into the optics by means of a fiber optic
bundle so that any mechanical vibrations of the wheel do not
interfere with the remainder of the system or create image
shifts.
As thus far described, the present invention images fluorescent
emissions having a polarization orthogonal to the polarization
state of the excitation light. This arrangement is suitable for
imaging samples which emit substantially depolarized emissions.
However, by incorporating a birefringent network between the PBS
element and the sample, it is additionally possible to image
fluorescent emissions having the same polarization as that of the
excitation light. In this implementation, the birefringent network
converts the polarization of the light in one wavelength band, such
as the emission light, to the orthogonal polarization state without
significant effect on the polarization of the other band, such as
the excitation light. Use of the birefringent network thus permits
imaging of either of the possible polarization states of the
emission light and, as hereinafter described, preserves the desired
rejection by the PBS element of stray excitation.
Any birefringent network may be used in such an embodiment of the
invention. The network may be as simple as a single optical
retarder (birefringent element), or may comprise a complex
arrangement including a plurality of retarders. When the network is
a single retarder, it is positioned in the beam between the PBS
element and the sample as shown in FIG. 7 (in which the
birefringent network is identified by reference numeral 55). The
retarder's fast axis is oriented at 45.degree. to the polarization
axis of the excitation light. Such an element has no effect on the
polarization of light for those wavelengths at which the waveplate
has integral order, i.e.
where R(.lambda.) is the retardance, .lambda. is the wavelength,
and m is an integer. However, the same retarder will convert the
polarization of light from one state to the orthogonal state at
wavelengths for which it exhibits half-integral order, i.e.
The retarder may for example comprise a fixed retarder formed of
polymers, quartz, calcite, or any other birefringent material.
Alternatively, the retarder may be a liquid crystal cell having a
fixed or a variable retardance. The retarder system may also be
formed of both fixed and variable retarders placed in optical
series. The choice of material will be determined by the particular
system, as will be apparent to persons of ordinary skill; exemplary
materials include nematic, smectic A*, and smectic C* materials,
quartz, calcite, LiNbO.sub.3, mylar, and optical retardation films
sold commercially by Polaroid, Sanritz, Nitto Denko, and
International Polarizer.
FIG. 6 graphically illustrates the functioning of a retarder. Curve
46 shows the transmission of un-altered polarized light. For a
single waveplate as referred to above, curve 46 exhibits maxima at
wavelengths at which the waveplate has integral order, and minima
at wavelengths for which it has half-integral order. Considering
the complementary case, the curve 47 -- depicting the efficiency of
transforming light from a selected polarization state to the
orthogonal state -- exhibits maxima at wavelengths for which the
waveplate has half-integral order, and minima at wavelengths for
which the waveplate has integral order. A second set of curves 46'
and 47' illustrate the corresponding properties of an exemplary
birefringent network of three retarders.
FIG. 7 depicts a system of the invention incorporating a
birefringent network 55 located optically between the PBS and
sample; the retardance network 55 converts substantially all of the
excitation light to its orthogonal polarization state with little
effect on the light emitted by the sample. Excitation light from
the PBS is polarized along an axis 51, and is converted by the
network 55 to the orthogonal polarization state 52. The converted
light 52 is directed to the objective 16 and then to the sample 20
from which it is reflected in the same state 52. As shown in FIG.
7, the reflected light is converted back to its original
polarization state 51 by the network 55, and is rejected at the PBS
15. Emission light from the sample in state 53 or 54, on the other
hand, proceeds through the network 55 without substantial
alteration of its polarization state. From network 55, the emission
light in state 54 is rejected at PBS 15 while emission light in
state 53 is transmitted by and through the PBS to form the
fluorescence image 24 on detector 25. This behavioral difference is
a result of the different excitation and emission wavelengths.
Thus, the retarder or retardance network 55 should be suitably
selected to accommodate the particular wavelengths involved -- i.e.
so that the retarder or network efficiently converts light from one
polarization state to the other at one wavelength band, but
converts (at most) little of the light at the other wavelength
band. The excitation light reflected from the PBS to the sample has
the "S" polarization state 51 which is converted by network 55 to
the "P" polarization state 52 and passed on to the sample. The
sample is thus illuminated in the "P" mode. Reflected light from
the sample has the "P" polarization state 52 which is converted
back to the "S" state 51 as it traverses network 55 a second time;
this S-mode light is reflected by the PBS and is thereby prevented
from continuing on to the imaging optics. When the sample and/or
fluorescent dye has even a partial orientation axis, it may be
desirable to image the sample in the "S" and/or "P" mode with
respect to the excitation and/or the emission light.
The emission light is not altered by the network 55. Since the PBS
transmits only the "P" polarized light 53, the fluorescent image is
formed by light which is emitted in the "P" polarized state; any
"S" polarized light emitted by the sample is rejected at the PBS.
Accordingly, the emission light imaged by the system has the same
polarization state as that of the excitation light. With the use of
a suitable birefringent network, the present invention is thus
effective at rejecting the excitation light so that it does not
interfere with the fluorescent image, even though the sample
emission has the same polarization state as the excitation
light.
In contrast, when no birefringent network is present, the
excitation light has "S" polarization while the emission light has
"P" polarization. Such a system images all randomly fluorescent
emissions.
The birefringent network works equally well if its action is
reversed. That is, the birefringent network can alternatively
transmit excitation light with an essentially unchanged
polarization state and transform the polarization of the emission
light from a selected state to the orthogonal state. This
modification is illustrated in FIG. 8 where a birefringent network
55' is interposed between the PBS 15 and the sample 20. Excitation
light from the PBS is "S" polarized, as indicated by reference
numeral 51, and is thus unaltered by the network 55'. The
excitation light proceeds to the sample, is reflected in the same
state 51, passes again through the network without significant
alteration, and is rejected at the PBS. Emission light in state 53
or 54 proceeds through the network 55' which converts light of
polarization state 53 to state 57 and light of polarization state
54 to state 58. Converted emission light in polarization state 57
is then rejected by the PBS, while converted light in state 58 is
transmitted by the PBS to form the fluorescence image 24 on
detector 25.
The excitation light is "S" polarized when it illuminates the
sample, and any reflected light is similarly "S" polarized. This
light is essentially unchanged as it passes through network 55' so
that it encounters PBS 15 as "S" polarized light and is reflected
at the PBS. "P" polarized sample emission is converted by the
network to the "S" state and is rejected at the PBS. Emissions from
the sample in the "S" state are converted by network 55' to the "P"
state and are transmitted through the PBS to form the fluorescent
image 24. Once again, the system is effective at rejecting unwanted
excitation light from the image, even though the sample emission
and sample excitation have the same polarization state.
In the preceding example, all beams have an "S" state at the
sample, while in the earlier example the beams are all "P"
polarized. Some sample species have an intrinsic polarization
sensitivity so that the polarization sense at the sample affects
the overall fluorescence intensity; an example of such a sample is
a fluorophore attached to a molecule which is bound to an oriented
membrane. In these circumstances, the sample has a preferred
polarization axis for optimum excitation and, for such samples,
there is an important difference of result whether a system of the
type shown in FIG. 7 (illumination with "P" mode light) or FIG. 8
(illumination with "S" mode light) is employed. Other systems in
accordance with the present invention and which permit measurements
along both the "S" and "P" polarization axes are described
below.
Because polarization state conversion depends on the birefringent
network to selectively convert the polarization state of either the
excitation light or the emission light, but not both, the same
birefringent network can be used for a variety of observations at a
variety of wavelengths. If the excitation light is chosen to have a
wavelength for which the network produces effectively no (or full)
conversion of a selected polarization state to its orthogonal
counterpart, and the emission light has a wavelength for which the
network produces full (or no) polarization conversion, then the
desired result will be obtained: the excitation light will be
rejected at the PBS, while the imaging emission light that has the
same polarization state at the sample that the excitation light had
at the sample will be transmitted.
The birefringent network may comprise a single retarder in any
system in which a narrow range of wavelengths is used for
excitation, such as when the light source is a laser. However, such
a network is unsatisfactory when a broader range of wavelengths is
used since such a network only achieves the ideal polarization
properties of complete conversion or completely unaltered
transmission at the exact wavelengths for which the retarder
exhibits, respectively, precisely half-integral or integral order.
When light having a range of wavelengths is used, the network will
produce a range of results, i.e. some unwanted transmission of
unconverted light and some unwanted conversion, thus reducing the
effectiveness of the system. Effective accommodation of a range of
wavelengths requires a multi-element network.
One suitable class of multi-element networks may be constructed
using the techniques described in a series of papers: "Optical
Network Synthesis Using Birefringent Crystals. I. Synthesis of
Lossless Networks of Equal-Length Crystals", J. Opt. Soc. Am.
54:1267 (1964) by S. E. Harris, E. O. Amman and I. C. Chang;
"Synthesis of Optical Birefringent Networks", Progress in Optics IX
(North-Holland, Amsterdam, 1971) pp. 123-177 by E. O. Amman; and
"Flat Passband Birefringent Wavelength Domain Multiplexer",
Electronics Letters 23(3), 106-7 (1987) by W. J. Carlsen and C. F.
Buhrer. The entire contents of these papers is incorporated by
reference. These papers describe synthesis of optical birefringent
Solc filters that have a prescribed passband and stopband, based on
a complex-exponential polynomial expansion similar to a Fourier
series. While the technique of Harris et al. is quite general, it
is sufficient here to consider a nominally lossless filter which
accepts and emits light polarized at 0.degree.. For this case, a
filter transmission of 1 corresponds to no conversion of
polarization state, while a transmission of zero corresponds to
complete conversion from a polarization state with an axis of
0.degree. to the orthogonal state with an axis of 90.degree..
For a mathematical description of the desired passband, the
synthesis procedure yields a number of orientation angles. The
filter is constructed by assembling a like number of retarders in
optical series, with their crystal axes oriented at
mathematically-specified orientation angles. Since the objective is
to obtain a wavelength-dependent polarization conversion rather
than a wavelength-dependent modulation of optical intensity, the
entrance and exit polarizers, which are normally used in a
birefringent filter to convert polarization state changes into
intensity changes, are omitted. Only the birefringent network is
required.
Carlsen and Buhrer describe the use of this technique to construct
filters with entrance and exit polarizations of 0.degree., with
equal-width passband and stopband, and exhibiting nearly a flat
response within each band with a periodic ripple of 1% or less.
This ripple magnitude is a design parameter analogous to the ripple
parameter used in the design of equiripple (Chebyshev) filters
well-known to electronics engineers. A filter constructed in
accordance with the Carlsen and Buhrer method with the intensity
converters omitted, is suitable for use in systems implementing the
present invention. The network should be oriented so that the
0.degree. axis (the nominal entrance polarizer axis) of the filter
is parallel to either the "S" or "P" state.
The result is a network with a set of maximally wide spectral bands
over which there is very little polarization conversion, and a
complementary set of bands over which there is substantially
complete polarization conversion. This provides a first set of wide
spectral regions over which to excite the sample, and a second set
of wide regions over which to collect the emitted light. Ideally,
the network should be designed so that each type of light,
excitation and emission, fits entirely within either a passband (no
conversion) or stopband (full conversion) of the network, with
little or no spill-over.
Such a network may for example be constructed in the manner shown
in FIG. 9, where the elements 61a and 61b are constructed so that
each has exactly twice the optical retardance of element 61c, and
the fast axes 62a-62c are oriented at angles of 80.1.degree.,
104.5.degree. and 45.degree., respectively. When retarder 61c is
chosen to have exactly one-half the retardance of elements 61a and
61b, the network yields a flat-topped response and the maximally
wide passband and stopband described above. The spectral location
of the passbands and stopbands are a function of the retardance
values of the elements 61a-c, as is well-known in the art. By way
of non-limiting example, quartz elements with thicknesses of 0.007"
for 61c and 0.014" for 61a and 61b may be used.
To selectively change the passbands and stop bands, a replaceable
birefringent network may be mechanically introduced and removed
from the beam between the PBS and the sample. It is preferable to
replace the network with another optical element having a
comparable thickness and optical refractive index to avoid focus
variations and image shifts when the network is switched in and out
of the optical path. The replacement element may be a second
birefringent network, a planar optical window, or any other optical
element.
FIG. 10 graphically illustrates the properties of a switchable
birefringent network for light of various wavelengths. At a first
setting of the switchable network, the network transmits light of a
specified polarization state, or converts it to the orthogonal
polarization state, depending on its wavelength. Spectral curves 70
and 72 illustrate (for this first switch state) the efficiency of
light transmission for a specified linear polarization of an
unaltered state and the efficiency of conversion into the
orthogonal state, respectively. For the other setting of the
switchable network, the network transmits polarized light of all
wavelengths without significant conversion into the orthogonal
polarization state. Spectral curves 74 and 76 depict for this other
switch setting the efficiency of light transmission for a specified
linear polarization in the unaltered state and the efficiency of
conversion into the orthogonal state, respectively.
In a preferred embodiment, the system includes an optical
polarization switch such as a liquid crystal cell which acts in
association with the birefringent elements. The switch has at least
one setting at which the overall optical effect of the birefringent
network is to transmit light within a specified range of
wavelengths without alteration of its polarization state.
Preferably, the switch has a second setting in which at the overall
optical effect is to convert light within a first selected
wavelength range from a selected polarization state to the
orthogonal polarization state, and to transmit light within a
second selected wavelength range without significant change to its
polarization state.
FIGS. 11a and 11b depict by way of example a suitable switch for
use in constructing a switchable birefringent network, in the form
of a variable optical retardance type liquid crystal cell which
exhibits approximately .lambda./2 retardance for light of a
specified wavelength range. It should be noted that FIGS. 11a and
11b are not to scale, and for purposes of clarity, show certain
very thin layers as enlarged. The cell 78 is shown in the
voltage-off state in FIG. 11a, and is shown and identified as 78'
in the voltage-on state in FIG. 11b. The cell comprises two
substrates 80 and 82 with transparent electrodes 81 and 83 on the
respective inner surfaces 87 and 88 thereof connected to a voltage
source 84. The layer or volume between the substrates 80 and 82 is
filled with a nematic liquid crystal material 86, oriented by
pretreatment of the inner surfaces of the substrates to have a
preferred molecular alignment direction at the two surfaces 87 and
88. Light passing through the cell has an optical axis 89.
Referring now to FIG. 12, a switchable birefringent network 100
comprising a first retarder network 108 of retarder elements 101a,
101b and 101c and having fast axes oriented at respective angles
102a, 102b, and 102c is shown with a liquid crystal switch 105 and
a further retardance network 109 comprising retarder elements 106a,
106b, and 106c having respective fast axes at angles 107a, 107b,
and 107c.
Switchable versions of birefringent networks are disclosed in
Miller and Buhrer's concurrently-filed co-pending patent
application entitled "Tunable Optical Filter with White State", the
entire contents of which are expressly incorporated by reference
herein. As described earlier herein, these networks may be
incorporated into the present invention to provide additional
versatility to the system. In one switch state, the network is
effectively absent from the optical path, whereas in the other
switch state the network functions in the manner of the
above-described non-switchable birefringent networks. The use of a
switchable network enables independent measurements of the sample
emissions to be readily obtained in either polarization state, so
that fluorescence polarization anisotropy and other measures of
interest may be assessed without need for any moving parts.
A fluorescent imaging system 110 constructed in accordance with the
invention and which incorporates a switchable birefringent network
115 interposed in the optical path between the PBS 15 and the
sample 20 is shown in FIG. 13. In that embodiment, excitation light
from PBS 15 is polarized along an axis 111. When the switch is in
one setting, the polarized light is converted by network 115 to the
orthogonal polarization state 112 and then proceeds through the
objective 16 to sample 20. At the sample, the light is reflected in
the same state 112, is converted back to state 111 by the network,
and is rejected at the PBS. Emission light in state 113 or 114, on
the other hand, proceeds through the network 115 without
substantial alteration to its polarization state. Emission light in
state 114 is rejected at the PBS while light in state 113 is
transmitted therethrough and forms the fluorescence image 24. When
the switch of the network 115 is in its other setting, the network
has substantially no effect on the polarization of either
excitation or emission light. As a consequence, unaltered "S"
polarized excitation light from the PBS proceeds through the
switchable network and illuminates the sample, from which any
reflections are "S" polarized and proceed, without alteration, back
through the network and are rejected at the PBS. Fluorescent
emissions from the sample pass without alteration through the
network 115; "S" polarized emissions are then rejected at the PBS
while "P" polarized emissions proceed through the PBS to the
imaging optics and form the fluorescent image 24. In summary, an
image obtained in the first switch state reveals the fluorescence
emissions having the same polarization state as the excitation. In
the other switch state, the image reveals the emissions polarized
orthogonally to the excitation light.
A suitable switchable birefringent network can be constructed
using, by way of example, 6 quartz elements oriented with their
fast axes at respective angles of -44, +22, -23.3, +113.3, +68 and
+134 degrees. Elements 2 and 5 have exactly twice the retardance of
the other elements. Positioned between the third and fourth quartz
elements is a liquid crystal variable retarder with an optical axis
oriented at 0.degree. and a retardance that is switchable between
approximately .lambda./2 and approximately 0 for light in the
wavelength range of interest. This may for example be achieved with
a nematic "pi" cell having substrates of Corning 7059 glass coated
with ITO on the inner surfaces to achieve a conductivity of 200
ohms/square, and spaced apart at a separation of 4 microns. The
region disposed between the substrates is filled with nematic
mixture ZLI-2772 (EM Industries, Hawthorne, N.Y.), which is free of
any chiral twist agents. The inner surfaces of the substrates are
buffed, in accordance with established methods in the art, to
produce a parallel (splay) orientation at 0.degree. with a pre-tilt
angle in the range of at least about 2.degree..
When a square-wave voltage of 25 Volts at 2 kHz is applied to the
cell, the overall network converts the polarization state of
certain wavelengths of light but not others, with approximately
equal portions of each, and a ripple of approximately 1% in the
passband (no conversion) and stopband (complete conversion) (FIG.
11b). When the voltage is removed, the liquid crystal molecules
re-orient in 1-2 milliseconds so that the overall network no longer
converts the polarization state of either excitation or emission
light. This state persists for approximately 40 ms, after which
time the liquid crystal cell undergoes a transition to a metastable
state shown in FIG. 11a. The unwanted effects of this transition
may be avoided by applying a "tickle" voltage or periodic "refresh"
signal to the cell, as is well-known in the art.
A twisted-nematic liquid crystal cell may also be interposed
between the PBS and the sample. Such a liquid crystal element
rotates the polarization state of light by the twist angle, which
is usually about 90.degree., and thereby affords a way to
selectively illuminate the sample with either "S" or "P" polarized
light. Engaging or energizing the cell also selects whether "S" or
"P" polarized emission light is measured at the detector. The
liquid crystal cell may be used in combination with the
above-described birefringent networks to select whether the
emission light being measured has the same polarization state as,
or the state orthogonal to, that of the excitation light.
In constructing a system which employs a twisted-nematic liquid
crystal cell, it is important that the cell produce the desired
rotation at both the excitation and emission wavelengths. While
there is generally some degree of variation with wavelength, the
variation may be minimized. One way to do so is to use a cell for
which the product of thickness and birefringence is large compared
to the wavelength of light, i.e. the product is at least 3.lambda..
This first approach relies on the principle of adiabatic following
and is highly successful over a broad spectral range. A cell with a
15 micron layer of the liquid crystal material ZLI-2772, having a
90.degree. helical twist, is for example suitable for use in the
visible range. Another approach is to use a cell that is operated
near the first or second Gooch-Tarry minimum for the specified
wavelength range. Such a cell is commonly well-known in the art and
is termed a "first-minimum" or "second-minimum" cell; sources of
these cells include Excel Technology (Belle Meade, N.J.) and
Standish Industries (Lake Mills, Wis.).
In the foregoing, several birefringent systems have been described
which vary the polarization state of the excitation light and/or
the emission light, without defeating the barrier provided by the
PBS against excitation light contributing to the fluorescent image.
It is possible to use several birefringent networks, or one or more
birefringent networks in series with a twisted nematic liquid
crystal cell, to combine the functions. This enables varying the
polarization state of the excitation light, the emission light, or
both. Also, several switchable birefringent networks may be
employed to provide operation at a variety of wavelengths.
Sequential mechanical engagement and disengagement of components,
or sequential application and removal of electrical signals to the
liquid crystal elements, may be used to produce sequential
illumination of the sample in each of the two orthogonal
polarization states and/or viewing of the sample in each of the two
orthogonal polarization states. This is useful in measurements of
fluorescence polarization.
Systems constructed in accordance with the present invention are
free of the NA restrictions inherent in the dark-field approach.
Thus, such systems are capable of high throughput in this regard,
i.e. use with high NA objectives such as the element 16 in FIG.
4.
Since polarized light is required for illumination but many light
sources do not inherently generate a polarized beam, the available
energy density at the sample using an unpolarized source is likely
to be no greater than about half that of an equivalent unpolarized
system. Accordingly, unless a polarized light source is available,
as may be provided with lasers, a prolonged and perhaps doubled
exposure period may be required to produce an equal amount of
fluorescent emission energy to unpolarized prior art systems.
On the other hand, "photobleaching" of the sample is also reduced
in the inventive systems by about one-half, so that longer exposure
periods generally will not cause a problem, except for the slower
image acquisition rate of the instrument. Furthermore, many current
instruments incorporate neutral-density filters to reduce the
excitation flux and reduce photobleaching. In these cases it is
therefore a straightforward procedure to reduce the amount of
otherwise-utilized neutral-density filtration to yield the same
excitation flux at the sample as prior art arrangements when using
a system of the present invention.
When it is critical that total excitation flux be maximized,
special techniques which preferentially convert the polarization
state of lamp-based sources so that the majority of the lamp flux
is in the desired polarization state may be employed. By way of
non-limiting example, a reflective polarizing film comprising a
thin polymeric substrate (in contrast to a coating on a glass
surface), together with a .lambda./4 waveplate to maximize total
excitation flux by means of converting one polarization state to
the other may be used. The film may be encapsulated in a glass or
plastic such as a polycarbonate. DBEF film from 3M Corporation, for
example, is a polarizer which transmits light of the desired
polarization state and reflects the unwanted state. The film is
placed after the .lambda./4 plate whose fast axis is oriented at
45.degree. to the DBEF polarization axis. Approximately 50% of the
incident light from an unpolarized source will be transmitted in
its first interaction with these elements. However, the remainder
of the incident light will not be transmitted but will, instead, be
reflected back towards the illumination optics through the
.lambda./4 waveplate. Some fraction of that light is reflected
again by the illumination optics back toward the film, passing a
second time through the .lambda./4 waveplate before again
encountering the DBEF film. Since two passes through a .lambda./4
waveplate will convert linearly polarized light to its
complementary state, any light rejected in the first attempt will
then be properly polarized to pass through the DBEF film.
To obtain best performance, the illumination system should
preferably be designed to control depolarized scatter and
reflections. Such a system design must insure that light reflected
by the DBEF film back into the illumination optics is not
obstructed by opaque elements such as the lamp arc or filament, or
by lamp supports and the like. The system is preferably designed to
permit multiple passages of the light without excessive losses to
beam divergence and vignetting. Properly implemented, arrangements
such as this can increase the excitation efficiency to well above
50%. Furthermore, since the DBEF film acts as an entrance polarizer
to the PBS, it notably improves the degree of polarization
extinction in that component.
Another concern is that since only half the fluorescent emission is
utilized (for a depolarized emission), at first glance the present
invention appears to utilize only half of the emission light of a
conventional epi-illumination system. However, many current imaging
systems employ filters or other elements which are polarization
sensitive and thus already sustain a similar loss. In other
instances, the loss in prior art systems is not inherent in the
measurement but arises from the use of an imaging technology that
is itself based on polarized light. A non-limiting list of such
polarization-sensitive elements includes diffraction gratings,
liquid crystal tunable filters (LCTFs), acousto-optic tunable
filters (AOTF's), surface plasmon filters, and cameras which
incorporate any of these elements.
More importantly, systems which utilize epi-illumination
beamsplitters with multiple excitation and emission bands are
rarely efficient in their use of emission light because of the
above-mentioned limitations of the dichroic beamsplitters. Often,
the emission band is not sufficiently broad, or is not
appropriately located to capture all of the fluorescent energy.
Such filtration losses rival, or exceed, the polarization losses in
systems of the present invention. Thus, in comparing losses in
multi-band systems, the epi-illumination approach is efficient in
terms of polarization but spectrally lossy (inefficient) because
the filtration of the beamsplitter is not spectrally well-matched
to the emission energy. On the other hand, the present invention
exhibits excellent spectral matching between the filters and the
emission energy. Which approach has higher throughput for a
particular application depends on which fluorescent species are
being imaged, how their spectra compare with the response of the
dichroic beamsplitter, and whether other elements necessary to the
system impose a polarization-selectivity of their own.
The present invention is ideally suited for use with
polarization-dependent elements such as LCTFs, AOTFs, and surface
plasmon filters. As compared with existing dichroic epi-illuminated
systems, systems of the present invention exhibit no loss in
efficiency and afford complete spectral freedom to excite and to
collect images at any desired wavelengths. These features make the
present invention ideal for multi-spectral imaging and imaging
spectroscopy.
In some cases, it may be desired to illuminate the sample through a
mechanical filter wheel. However, having eliminated mechanical
motion in the remainder of the system, it is not desirable to
introduce vibrations through the filter wheel. Thus, it is often
beneficial to locate such a filter wheel separately from the
remainder of the system and to couple the excitation light into the
PBS by means of fiber and associated optics.
FIG. 14 depicts such an embodiment of the invention in which the
illumination system includes a fiber-optic feed 90. As there shown,
a filter wheel 91 holding filters 92a, 92b, and 92c is used to
select the excitation wavelength. Wheel 91 is located separate from
the remainder of the system so as to eliminate vibration and image
distortions as the excitation wavelength is changed. While a fiber
optic system can thus be used to direct the excitation light
(inasmuch as there is no need to preserve a spatially coherent
image of the light source when illuminating the sample), a fiber
optic system may not be employed in the emission path since this
would degrade, or destroy, the spatial information forming or
comprising the image of the sample. By providing a high-quality
optical path for the emission light free of mechanical motion and
vibration, the present invention is distinct in purpose from prior
art arrangements and cannot utilize a fiber optic system on the
emission or imaging side.
The imaging optics direct the emission light to a detector which
views the fluorescent image. It is possible to use nearly any
detector 25 with suitable signal-to-noise and detection
performance. A non-limiting list of suitable detectors includes the
human eye; photographic film; a CCD camera (such as the Model
Micromax KAF-1400 from Princeton Instruments); a vidicon tube; an
image intensifier tube or microchannel plate; and an avalanche
photodiode, photomultiplier tube, or photodiode when only a single
spatial reading is required.
FIG. 15 shows a system in accordance with the present invention
having a tunable filter or spectrometer 120 disposed in the
emission path. FIG. 16 shows still another system of the invention
including a filter wheel or tunable filter in the illumination path
and a plurality of birefringent networks 131a, 131b, and 131c
between the PBS 15 and sample 20.
Although it is within the intended scope of the invention to
construct an imaging system using individual components and
elements, it is more economical to construct or implement the
system using an existing fluorescence microscope, such for example
as the Axiovert 135 or Axioskop made by Zeiss (Jena, Germany), or
the BX 60 from Olympus, or the E 800 from Nikon. These existing
microscopes conveniently provide suitable illumination and imaging
optics with high efficiency and good optical quality.
Conventional fluorescence microscopes include a structure for
incorporating a dichroic reflector that is typically mounted on a
slider or other removable member. The dichroic reflector may be
replaced in accordance with the invention with a PBS that is
dimensionally compatible with the available space.
The PBS may be a cube design, a plate design, or a
double-refraction device such as a Rochon or Glan-Taylor prism.
Another alternative is to use a DBEF film as the PBS. DBEF is
typically provided in sheet form, and may be laminated between
high-quality glass flats of e.g. BK-7 from Schott (Duryea, Pa.) by
means of optical cement or epoxy such as UV-15 from MasterBond
(Hackensack, N.J.). The exterior glass faces should be
anti-reflection coated so that Fresnel reflections do not interfere
with, or destroy, the otherwise high degree of polarization
efficiency.
The degree of selectivity provided by the PBS alone may not be as
high as desired. However, the selectivity of the instrument can be
improved by a system of the invention constructed as shown in FIG.
17.
FIG. 17 depicts an imaging system 170 that further incorporates a
polarizing element 171 which at least partially polarizes the
excitation light 13 incident upon the PBS 15. The polarizing
element 171 may optionally comprise a reflective polarizer element
or, in another embodiment, a reflective polarizer element
incorporating a .lambda./4 wave plate 175. Polarizing element 172
in the emission path at least further polarizes the partially
polarized emission light that exits the PBS and is incident upon
the element 172. These elements further perfect the polarizing
action of the PBS.
In yet another embodiment a reflector 174 is located to direct
light reflected by the reflective polarizing element back towards
the reflective polarizing element. Reflector 174 can be behind the
light source or incorporated as part of the light source
housing.
In the embodiment illustrated in FIG. 17, the first location for a
polarizer, identified as 171, is at the entrance face at which the
excitation light is introduced to the PBS 15. First polarizing
element 171 ensures that only "S" mode linearly polarized light is
presented to the PBS and that any imperfections at the first
interaction between the excitation light and the PBS will introduce
only a loss in efficiency rather than a reduced polarization
purity. The second location for a polarizing element, identified as
172, is at the exit face of the PBS 15 for admitting light from the
PBS to the imaging optics 23. A linear polarizer 172 so placed
removes stray "S" polarized light from the emission beam and
increases the polarization purity of the PBS for transmitted "P"
light. Performance limitations whereby some "P" light is reflected
at the PBS hypotenuse do reduce the efficiency of collection for
emission light, but this is generally an acceptably small amount on
the order of 1% or less; this emission light is reflected into the
illumination optics, where it should not normally cause a
problem.
In implementing systems in accordance with the invention, any
polarizer which operates in the required spectral range may be
used. A nonlimiting list of such components includes commercial
sheet dichroic polarizers such as: HN32, HN-38S or HN42 from
Polaroid (Norwood, Mass.) and similar products for the visible
range; HNP'B for the UV range and HR for the near-infrared, both
from Polaroid; calcite prism polarizers such as the model RA-10-10
Rochon polarizer from Karl Lambrecht (Chicago, Ill.); and DBEF
material from 3M Corporation. The latter class of materials is best
operated either at normal incidence or at an off-normal angle, with
the optimum choice depending on the overall system design.
The near UV ranges may be important for the excitation of
particular fluorescent dye species. The excitation range may be
extended by taking advantage of the fact that the PBS need only
afford reflection, not true polarization separation, at short
wavelengths which are used only for excitation and not for
emission. The PBS designer can then sacrifice polarization purity
in the UV range, and seek only high reflection. Such UV light
reflected from the sample is not removed from the beam by the PBS,
as would normally be the case in utilizing the present invention,
but may instead be accommodated by placing a UV cut-on glass filter
as the first element in the imaging optics. Suitable PBS units
operable in the UV range may by way of example be obtained from
Karl Lambrecht of Chicago, Ill. For systems which image only
visible emissions, one suitable glass filter is GG-400 from Schott
(Duryea, Pa.). This cut-on filter must have a high transmission at
all wavelengths for which emissions are to be imaged, and the PBS
must also exhibit at those wavelengths its normal properties of
polarization selectivity.
The present invention is not limited to use in the visible
wavelength range and, throughout this disclosure, the term light is
intended to indicate and include optical radiation of the visible,
ultraviolet, and infrared ranges. The inventive systems may be
constructed in many ways, and its components may be assembled in
various combinations and with such substitutions of materials,
thicknesses, angles, components and other aspects as will be
evident to those skilled in the art as embodying rather than
deviating from the invention described herein.
Thus, while there have shown and described and pointed out
fundamental novel features of the invention as applied to a
preferred embodiment thereof, it will be understood that various
omissions and substitutions and changes in the form and details of
the devices illustrated, and in their operation, may be made by
those skilled in the art without departing from the spirit of the
invention. For example, it is expressly intended that all
combinations of those elements and/or method steps which perform
substantially the same function in substantially the same way to
achieve the same results are within the scope of the invention.
Moreover, it should be recognized that structures and/or elements
and/or method steps shown and/or described in connection with any
disclosed form or embodiment of the invention may be incorporated
in any other disclosed or described or suggested form or embodiment
as a general matter of design choice. It is the intention,
therefore, to be limited only as indicated by the scope of the
claims appended hereto.
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